Gastroenteritis is a worldwide infectious disease problem that is the major cause of neonatal and infant mortality in developing countries and of significant morbidity in developed countries. Recognition that rotaviruses are the major viral pathogens that cause life-threatening diarrheal disease has focused vaccine development against these pathogens. Unfortunately, intense effort by others to develop an effective live oral vaccine has not yet resulted in a successful vaccine, and it has failed to provide definitive information on what constitutes a protective immune response. Our previous work developed a new animal model in rabbits to monitor active immunity against rotavirus infection and we successfully developed rotavirus-like particles (VLPs) composed of capsid proteins expressed in high yield and assembled in insect cells. We showed for the first time that parenteral vaccination with nonreplicating rotavirus (inactivated virus or VP2/4/6/7 VLPs) can induce active protection in the rabbit model. This grant proposes to extend these initial exciting observations. We will define the minimal viral proteins needed to induce homotypic as well as heterotypic immunity using a VLP subunit vaccine, and optimize a vaccination regimen to effectively induce mucosal immunity. Vaccine formulation (VP2/4/6/7, VP2/4/6, VP2/6/7,VP2/6 and mosaic VLPs with proteins from different serotypes), vaccine dose, and different types of adjuvant and delivery systems for oral or parenteral immunization (or a combination of these routes) will be evaluated for their effectiveness in stimulating both mucosal and systemic immunity and protection from virus challenge. The ability of each vaccine/adjuvant combination to stimulate mucosal immunity will be dissected by basic studies of the B and T cell responses in mice, and will provide critical information on how to optimize a mucosal response. These basic studies will be performed in collaboration with the Mucosal Immunization Research Group at the University of Alabama. Following the immunogenicity studies, active protection will be tested in a rabbit model and in a mouse model. Our long-term goal is to develop a subunit rotavirus vaccine to be given orally to children. Results from our proposed research will provide insight on protective immunity to rotavirus and directly assess the feasibility of using VLPs as vaccines. Our studies also will serve as a model for general applicability for the use of nonreplicating subunit vaccines from other enteric pathogens as mucosal vaccines. Results from this research potentially have broad applicability for enhancement of the immunogenicity of subunit vaccines (including HIV), and for oral delivery or targeting of drugs, including antiviral and anticancer agents, to the gastrointestinal tract.